Peptidyl nucleosides (PNs) are naturally occurring antifungal agents active against multiple pathogenic fungi such as the causal agent of Valley Fever. They also exhibit potent synergistic effects with clinically approved antifungal drugs. Our long-term goal is to provide a comprehensive understanding of both the biosynthesis of PNs and their mode of action. The current application focuses on the biosynthesis of PNs. Understanding the PN biosynthetic pathways will provide a basis for creating structurally diverse PN analogs through engineered biosynthesis, semi synthesis and genome mining. In this application, we will establish the common steps in PN biosynthesis by testing the central hypothesis that the structural features common to all PNs (aminohexuronic acid and its amide linkage to an amino acid) are biosynthesized through the action of seven conserved enzymes. In Aim 1, NikJ, a newly identified free radical dependent enzyme responsible for the synthesis of a key cyclic nucleotide intermediate, will be mechanistically characterized. In Aim 2, NikS, the enzyme responsible for the ligation of the N-terminal amino acid to nucleoside (aminohexuronic acid), will be characterized for both its substrate specificity and its potential for use in the chemoenzymatic preparation of PNs. In Aim 3, the enzymes responsible for aminohexuronic acid formation will be identified through gene knockout experiments. The proposed research is significant because it will provide a basis for the future biosynthetic and chemoenzymatic generation of novel therapeutic PNs.